Stable liquid formulation of human growth hormone

ABSTRACT

Disclosed herein is a stable liquid formulation comprising human growth hormone; L-lysine, L-arginine or polyethylene glycol 300; and poly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate or polyethylene glycol-35 castor oil.

TECHNICAL FIELD

The present invention relates to a stable liquid formulation comprisinghuman growth hormone; L-lysine, L-arginine or polyethylene glycol 300;and poly(oxyethylene) poly(oxypropylene) copolymer, polyethyleneglycol-15 poly-oxystearate or polyethylene glycol-35 castor oil.

BACKGROUND ART

Human growth hormone, which is produced in the human pituitary gland, isa single-chain polypeptide of 191 amino acids having a molecular weightof about 22,000 daltons. Human growth hormone is used mainly fortreating pituitary dwarfism in children (Endocrinol. Rev. 4, 155, 1983).

Human growth hormone tends to be unstable in pharmaceuticalpreparations. The degradation products of growth hormone, includingdeamidated or sulfoxylated products and dimer or polymer forms, can begenerated in solution. A predominant chemical degradation reaction ofhuman growth hormone is deamidation, which especially takes place at theasparagine residue at position 149, by direct hydrolysis or via a cyclicsuccinimide intermediate to form L-asp-hGH, L-iso-asp-hGH, D-asp-hGH andD-iso-asp-hGH. At present, these deamidated products of human growthhormone are not believed to have toxic or altered biological activity orreceptor binding properties, but the conformational stability of thesulfoxides is reduced compared to native human growth hormone.

The deamidated human growth hormone is undesirable for use as amedicament because it undergoes quality deterioration, despite havingunaltered biological activity, and its allowable content is thus usuallyprovided by the public specification for purity. Also, the formation ofaggregates of growth hormone, such as dimers or polymers, causesundesired immunogenicity, leading to a safety problem in vivo, andsuspended solids, leading to an appearance problem, thereby causingpatients displeasure.

Protein stability is closely related with water. Human growth hormone ismostly commercially available in a lyophilized formulation to increaseits stability that must be reconstituted prior to use. However, manystudies have been conducted on liquid formulations of human growthhormone in order to improve the convenience of medical doctors andpatients. For example, U.S. Pat. No. 6,448,225, relating to Genentech'sNutropin AQ, discloses an aqueous formulation of human growth hormone,comprising human growth hormone, mannitol, a buffer and a non-ionicsurfactant, wherein citrate buffer was exemplified as being preferable.Korean Pat. Application No. 10-1999-0001217 provides a stable aqueousformulation, in which human growth hormone is dissolved in a buffersolution, which is prepared using a slightly to moderately acidicbuffer, preferably a maleate buffer, containing benzalkonium chloride.Korean Pat. Application No. 10-1998-0052483 describes apharmaceutically-stable liquid formulation of human growth hormone,wherein a buffer solution consisting of sodium acetate and sodiumglutamate is preferred. Also, in Korean Pat. Application No.10-1994-0702139, Novo Nordisk A/S describes a pharmaceutical formulationcomprising a growth hormone, and an amino acid selected from the groupconsisting of Asp, Ile, Val, Leu and His, or a derivative of histidine,or a peptide comprising at least one basic amino acid and at least oneacidic amino acid, and a non-ionic detergent, such as polysorbate orpoloxamer. Novo Nordisk A/S currently markets Norditropin SimpleXx,which contains human growth hormone, histidine buffer, a non-ionicsurfactant, sodium chloride, and a preservative.

However, although a variety of attempts have been made to provide anliquid formulation of growth hormone, as described above, there is aneed for a stabler liquid formulation of growth hormone.

DISCLOSURE OF INVENTION Technical Problem

It is therefore an object of the present invention to provide a stableliquid formulation of human growth hormone by combining a specific aminoacid, such as L-lysine or L-arginine, or polyethylene glycol, with aspecific surfactant in order to minimize the formation of deamidatedproducts and aggregation, thereby enhancing long-term storage stabilityand convenience relative to known liquid formulations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the residual rate (%) of human growth hormoneaccording to deamidation and aggregation when a liquid formulationaccording to the present invention is stored at 25° C. for three months.

FIG. 2 is a graph showing the residual rate (%) of human growth hormoneaccording to deamidation and aggregation when a liquid formulationaccording to the present invention is stored at 2° C. to 8° C. for oneyear.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention relates to a stable liquidformulation comprising human growth hormone; L-lysine or L-arginine; andpoly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15polyoxystearate or polyethylene glycol-35 castor oil.

Human growth hormone, which is intended to be stabilized in the liquidformulation of the present invention, may be naturally occurring, or maybe obtained from a prokaryote or eukaryote transformed with DNA codingfor human growth hormone using a recombinant DNA technique. A method ofproducing human growth hormone using E. coli or yeast as a generecombinant host is described in the literature, for example, KoreanPat. No. 25013, Korean Pat. No. 316347, or Korean Pat. Publication No.10-1997-0006498. The human growth hormone may be the wild type or aderivative thereof.

The liquid formulation of the present invention comprises L-lysine orL-arginine as a stabilizer. Preferred is a salt form of the stabilizer,for example, L-lysine hydrochloride or L-arginine hydrochloride. Thestabilizers exhibited a good effect of causing no aggregation afterstorage for a predetermined period of time in the liquid formulation ofhuman growth hormone compared to other conventional amino acidstabilizers. The aforementioned stabilizers, as shown in Table 8,underwent no aggregation after 14 days, and thus displayed goodstability compared to histidine, which is employed in International Pat.Publication No. WO 1997/39768. In the provision of a stable liquidformulation of human growth hormone, these results exhibit a good effectthat was not disclosed in the aforementioned cited patent, disclosingonly the use of an amino acid as a stabilizer, and other knownliterature.

The poly(oxyethylene) poly(oxypropylene) copolymer (poloxamer),macrogol-15 polyoxystearate (polyethylene glycol-15 polyoxystearate) orcremophor ELP (polyethylene glycol-35 castor oil), used in the liquidformulation of the present invention, is a non-ionic surfactant. Whenthe present inventors employed the aforementioned surfactants, amongvarious non-ionic surfactants, to combine them with a specificstabilizer, the liquid formulation was found to have enhanced stability.These surfactants displayed good effects with respect to the deamidationand aggregation of human growth hormone compared to conventionalpolysorbates, such as Tween 20 or Tween 80 (Table 5). The poloxamer ispreferably poloxamer 188 (poly(oxyethylene) poly(oxypropylene) copolymer188) or poloxamer 407 (poly(oxyethylene) poly(oxypropylene) copolymer407), and more preferably poloxamer 188 (poly(oxyethylene)poly(oxypropylene) copolymer 188).

The liquid formulation of the present invention may further include anadditive known in the art, for example, a buffer, an isotonic agent, apreservative or an analgesic.

The buffer may be used to adjust the pH value of the liquid formulationwith no effect on human growth hormone, and is exemplified by acetate,glutamate, lactate, malate, citrate and phosphate. Sodium citrate orsodium acetate is preferred. As is apparent from the results of Example4, the buffer does not affect the stability of the liquid formulation ofthe present invention. These results are obtained because the goodstability of the liquid formulation of the present invention resultsfrom the combination of a specific stabilizer and a specific surfactant.The buffer is present at a concentration of 5 to 100 mM, and preferably5 to 50 mM, in the liquid formulation of the present invention.

The isotonic agent useful in the present invention may include sodiumchloride, mannitol, sucrose, dextrose, sorbitol, or mixtures thereof.D-mannitol is preferred. The isotonic agent is preferably present at aconcentration of 20 to 50 mg/ml in the liquid formulation of the presentinvention.

The preservative useful in the present invention may include benzylalcohol, phenol, and meta-cresol. Benzyl alcohol is preferred. Thebenzyl alcohol serves as an analgesic as well as a preservative. Thepreservative is preferably present in an amount ranging from 1 to 9mg/ml in the present liquid formulation.

In a detailed aspect, the liquid formulation of the present inventioncomprises 2.5 to 7.5 mg/ml of human growth hormone 0.01 to 1.0% (w/v) ofL-lysine or L-arginine per D of human growth hormone and 0.01 to 1.0%(w/v) of poly(oxyethylene) poly(oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethylene glycol-35 castor oil. In afurther detailed aspect, the liquid formulation comprises 2.5 to 5.5mg/ml of human growth hormone; 0.02 to 0.5% (w/v) of L-lysine orL-arginine per mg of human growth hormone; and 0.1 to 0.5% (w/v) of poly(oxy ethylene) poly(oxypropylene) copolymer, polyethylene glycol-15polyoxystearate or polyethylene glycol-35 castor oil.

In a preferred aspect, the liquid formulation of human growth hormoneaccording to the present invention comprises 2.5 to 5.5 mg/ml of humangrowth hormone; 0.02 to 0.5% (w/v) of L-lysine or L-arginine per D ofhuman growth hormone; 0.1 to 0.5% (w/v) of poly(oxyethylene)poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate orpolyethylene glycol-35 castor oil 5 to 20 mM of sodium citrate or sodiumacetate; 20 to 50 mg/ml of D-mannitol; and 1 to 9 mg/ml of benzylalcohol. The liquid formulation is moderately acidic or neutral, andpreferably has a pH value between 5.8 and 7.0, and more preferably a pHvalue between 6.0 and 6.2.

In another aspect, the present invention relates to a stable liquidformulation comprising human growth hormone; polyethylene glycol 300;and poly(oxyethylene) poly(oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethylene glycol-35 castor oil.

The human growth hormone and the poly(oxyethylene) poly(oxypropylene)copolymer, polyethylene glycol-15 polyoxystearate or polyethyleneglycol-35 castor oil contained in the liquid formulation are the same asdescribed above.

The liquid formulation of the present invention may include polyethyleneglycol 300 as a stabilizer. Polyethylene glycol is a useful polymer thatis water-soluble and dissolved in various organic solvents. Sincepolyethylene glycol is non-toxic and rapidly cleared from the body, itis used for a stable pharmaceutical composition (International Pat.Publication No. WO 01/26692). Korean Pat. Application No.10-2003-0061434 discloses a liquid formulation of human growth hormone,comprising polyethylene glycol instead of polysorbate as a non-ionicsurfactant, and not containing an antiseptic such as benzyl alcohol orphenol. However, this application describes the use of 0.001 to 20 mg/mlof polyethylene glycol having a molecular weight higher than 3,000 Daamong polyethylene glycols, without using a surfactant. This is incontrast to the present invention, which employs polyethylene glycolhaving a molecular weight of 300 Da, i.e., polyethylene glycol 300,along with a specific surfactant in order to improve the stability ofthe liquid formulation. The present invention reduces aggregationthrough the combinational use of a specific polyethylene glycol and aspecific surfactant, as described above, thereby enabling the long-termstorage of a liquid formulation.

The above liquid formulation of the present invention may furtherinclude an additive known in the art, for example, a buffer, an isotonicagent, a preservative or an analgesic. The buffer, isotonic agent,preservative or analgesic is the same as described above.

In a detailed aspect, the liquid formulation of the present inventioncomprises 2.5 to 7.5 mg/ml of human growth hormone; 0.1 to 5.0% (v/v) ofpolyethylene glycol 300 per mg of human growth hormone; and 0.01 to 1.0%(w/v) of poly(oxyethylene) poly(oxypropylene) copolymer, polyethyleneglycol-15 polyoxystearate or polyethylene glycol-35 castor oil. In afurther detailed aspect, the liquid formulation comprises 2.5 to 5.5mg/ml of human growth hormone; 0.2 to 1.0% (v/v) of polyethylene glycol300 per mg of human growth hormone; and 0.1 to 0.5% (w/v) ofpoly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15polyoxystearate or polyethylene glycol-35 castor oil.

In a preferred aspect, the liquid formulation of human growth hormoneaccording to the present invention comprises 2.5 to 5.5 mg/ml of humangrowth hormone; 0.2 to 1.0% (v/v) of polyethylene glycol 300 per mg ofhuman growth hormone; 0.1 to 0.5% (w/v) of poly(oxyethylene)poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate orpolyethylene glycol-35 castor oil 5 to 20 mM of sodium citrate or sodiumacetate; 20 to 50 mg/ml of D-mannitol; and 1 to 9 mg/ml of benzylalcohol. The liquid formulation is moderately acidic or neutral,preferably has a pH value between 5.8 and 7.0, and more preferably has apH value between 6.0 and 6.2.

MODE FOR THE INVENTION

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

Example 1 Preparation and Analysis of a Liquid Formulation of HumanGrowth Hormone

A liquid formulation according to the present invention was prepared asfollows. 4.0 mg/ml of native human growth hormone (Daewoong, Korea),which was provided in a bulk solution prior to being lyophilized, weremixed with a 10 mM aqueous solution of sodium acetate (Sigma), sodiumcitrate (Sigma) or sodium phosphate monobasic (Sigma) as a buffer so asto be adjusted to a pH ranging from 6.0 to 6.2. After the solution wasadjusted to the final concentration, it was mixed with an excipient(D-mannitol, Sigma), a surfactant {macrogol-15 polyoxystearate (SolutolHS 15, BASF), poloxamer 188 (Lutrol F 68, BASF), poloxamer 407 (Lutrol F127, BASF), cremophor ELP (BASF), Tween 20 (CRILLET 1 HP, Croda) orTween 80 (CRILLET 4 HP, Croda)}, a stabilizer (polyethylene glycol 300(PEG 300, BASF), polyethylene glycol 400 (PEG 400, BASF), PVPK-12(Kollidon 12 PF, BASF), PVPK-15 (Kollidon 15 PF, BASF), L-lysine(L-Lysine-HCl, Sigma) or L-arginine (L-Arginine-HCl, Sigma)), and benzylalcohol (Daejung, Korea) as a preservative.

The resulting liquid formulation was stored under strict conditions of40° C. and 75% RH for two weeks. The formulation was examined for thestability of human growth hormone according to deamidation andpolymerization over time. Deamidation was determined using reverse-phaseHPLC (RP-HPLC), and the formation of dimers and polymers by sizeexclusion HPLC (SEC-HPLC), according to the European Pharmacopoeiamethod (EP method).

Example 2 Evaluation of Effects of Surfactants on the Stability of HumanGrowth Hormone

In order to investigate the effects of pharmaceutically acceptablesurfactants on the stability of human growth hormone, liquidformulations of human growth hormone were prepared using the componentslisted in Table 1 and then analyzed according to the same method as inExample 1.

TABLE 1 Major Isotonic Component Buffer Agent Surfactant PreservativeSolution Control Somatropin Sodium D-mannitol — Benzyl Injectable 3.3 mgacetate 10 mM 50 mg alcohol 2.5 mg solution1 ml DWF 1 Somatropin SodiumD-mannitol Tween 202 mg Benzyl Injectable 3.3 mg acetate 10 mM 50 mgalcohol 2.5 mg solution1 ml DWF 2 Somatropin Sodium D-mannitolMacrogol-1 Benzyl Injectable 3.3 mg acetate 10 mM 50 mg 5 poly- alcohol2.5 mg solution1 ml oxystearate 2 mg DWF 3 Somatropin Sodium D-mannitolCremophor Benzyl Injectable 3.3 mg acetate 10 mM 50 mg ELP2 mg alcohol2.5 mg solution1 ml DWF 4 Somatropin Sodium D-mannitol Tween 802 mgBenzyl Injectable 3.3 mg acetate 10 mM 50 mg alcohol 2.5 mg solution1 mlDWF 5 Somatropin Sodium D-mannitol Poloxamer Benzyl Injectable 3.3 mgacetate 10 mM 50 mg 1882 mg alcohol 2.5 mg solution1 ml

The liquid formulations thus prepared were evaluated for the deamidationand aggregation of human growth hormone according to the type ofsurfactants, and the results are given in Table 2, below.

In Table 2, deamidation (%) indicates the calibrated residual rate (%)of non-deamidated active human growth hormone, and aggregation indicatesthe calibrated residual rate (%) of active human growth hormone notforming dimers or polymers, according to time at 40° C.

TABLE 2 DWF2 (Macrogol- DWF3 DWF5 Control DWF 1 15 poly- (cremophor DWF4(poloxamer (—) (Tween20) oxystearate) ELP) (Tween 80) 188) DeamidationDay 8 65.2 ± 0.5 81.9 ± 0.3 83.1 ± 0.3 83.4 ± 0.2 82.7 ± 0.2 83.1 ± 0.3(SomatropIn Day 45.5 ± 0.4 69.1 ± 0.2 68.5 ± 0.4 69.1 ± 0.2 68.9 ± 0.269.6 ± 0.2 %) 14 Aggregation Day 8 98.0 ± 0.2 100.0 ± 0.0  100.0 ± 0.0 100.0 ± 0.0  99.9 ± 0.1 100.0 ± 0.0  (Somatropin Day 80.2 ± 0.3 99.4 ±0.1 99.1 ± 0.1 99.6 ± 0.1 99.7 ± 0.1 99.5 ± 0.1 %) 14

Compared to the control, formulations preparing using surfactants werefound to suppress deamidation and aggregation. These results indicatethat the use of a surfactant improves the stability of a liquidformulation of human growth hormone.

Example 3 Evaluation of Effects of Stabilizers on the Stability of HumanGrowth Hormone

In order to investigate the stability of a liquid formulation of humangrowth hormone according to stabilizers, liquid formulations of humangrowth hormone were prepared using the components listed in Table 3 andthen analyzed according to the same method as in Example 1.

TABLE 3 Major Isotonic component Buffer agent Surfactant StabilizerPreservative Solution Control Somatropin Sodium D-mannito Tween — BenzylInjectable 3.3 mg acetate 150 mg 202 mg alcohol 2.5 mg Solution 1 ml 10mM DWF 6 Somatropin Sodium D-mannito Tween PEG Benzyl Injectable 3.3 mgacetate 150 mg 202 mg 3001% alcohol 2.5 mg Solution 1 ml 10 mM v/v DWF 7Somatropin Sodium D-mannito Tween PEG Benzyl Injectable 3.3 mg acetate150 mg 202 mg 4001% alcohol 2.5 mg Solution 1 ml 10 mM v/v DWF 8Somatropin Sodium D-mannito Tween PVP K- Benzyl Injectable 3.3 mgacetate 150 mg 202 mg 125 mg alcohol 2.5 mg Solution 1 ml 10 mM DWF 9Somatropin Sodium D-mannito Tween PVP K- Benzyl Injectable 3.3 mgacetate 150 mg 202 mg 175 mg alcohol 2.5 mg Solution 1 ml 10 mM DWF 10Somatropin Sodium D-mannito Tween L-Lys•HCl 1 mg Benzyl Injectable 3.3mg acetate 150 mg 202 mg alcohol 2.5 mg Solution 1 ml 10 mM DWF 11Somatropin Sodium D-mannito Tween L-Lys•HCl 1 mg Benzyl Injectable 3.3mg acetate 150 mg 202 mg alcohol 2.5 mg Solution 1 ml 10 mM

The liquid formulations thus prepared were estimated for deamidation andaggregation according to the type of stabilizers, and the results aregiven in Table 4, below.

TABLE 4 Deamidation Aggregation (somatropin %) (somatropin %) Day 7 Day14 Day 7 Day 14 Control (-) 70.8 ± 0.2 52.0 ± 0.3 100.0 ± 0.0 97.0 ± 0.2DWF6 (PEG 83.7 ± 0.1 70.6 ± 0.2 100.0 ± 0.0 99.6 ± 0.1 300) DWF7 (PEG79.7 ± 0.1 66.9 ± 0.1 100.0 ± 0.0 99.3 ± 0.1 400) DWF8 (PVP 80.8 ± 0.269.0 ± 0.2 100.0 ± 0.0 99.5 ± 0.1 K-12) DWF9 (PVP 80.7 ± 0.2 69.0 ± 0.2100.0 ± 0.0 99.4 ± 0.1 K-17) DWF10 (L- 83.3 ± 0.1 70.4 ± 0.1 100.0 ± 0.0100.0 ± 0.0  Lys□HCl) DWF11 (L- 83.5 ± 0.1 70.2 ± 0.1 100.0 ± 0.0 100.0± 0.0  Agn□HCl)

As shown in Table 4, among the stabilizers that were used, PEG 300(polyethylene glycol 300) and two amino acids, L-Lys

HCl and L-Arg

HCl, were found to stabilize human growth hormone. In particular, whenL-lysine and L-arginine were used, aggregation was suppressed to arelatively high degree in a formulation.

Example 4 Evaluation of Effects of Buffers on the Stability of HumanGrowth Hormone

The liquid formulation containing PEG 300, which was proven to stabilizehuman growth hormone in a liquid formulation in Example 3, was furtherexamined for the stability of human growth hormone according to the typeof buffers.

Liquid formulations of human growth hormone were prepared from thecomponents listed in Table 5 according to the same method as in Example1.

TABLE 5 Major Isotonic component Buffer agent Surfactant StabilizerPreservative Solution DWF12 Somatropin Sodium D-mannito Tween PEG BenzylInjectable 3.3 mg acetate 150 mg 202 mg 3001% alcohol 2.5 mg solution 1ml 10 mM v/v DWF13 Somatropin Sodium D-mannito Tween PEG BenzylInjectable 3.3 mg citrate 10 mM 150 mg 202 mg 3001% alcohol 2.5 mgsolution 1 ml v/v DWF14 Somatropin Sodium D-mannito Tween PEG BenzylInjectable 3.3 mg phosphate 150 mg 202 mg 3001% alcohol 2.5 mg solution1 ml 10 mM v/v

The stability of the liquid formulations thus prepared according to thetype of buffers was determined by analyzing the formation of deamidatedforms and dimers/polymers of human growth hormone over time at 40° C.The residual rates of active human growth hormone were described inTable 6, below.

TABLE 6 Deamidation Aggregation (somatropin %) (somatropin %) Day 7 Day14 Day 7 Day 14 DWF12 83.7 ± 0.1 70.6 ± 0.2 100.0 ± 0.0 99.27 ± 0.3(sodium acetate) DWF13 82.7 ± 0.5 70.9 ± 0.1 100.0 ± 0.0 99.62 ± 0.1(sodium citrate) DWF14 80.8 ± 0.4 69.0 ± 0.3 100.0 ± 0.0 99.12 ± 0.4(sodium phosphate)

The buffers were conventionally described as being important in a liquidformulation of human growth hormone in the aforementioned patent andapplications (U.S. Pat. No. 6,448,225, Korean Pat. Application No.10-1999-0001217, and Korean Pat. Application No. 10-1998-0052483).However, the liquid formulation of the present invention, as shown inTable 6, exhibited no marked difference in stability depending to thetype of conventionally used buffer.

Example 5 Comparison of Stability of Liquid Formulations

In order to compare the stability of liquid formulations according tothe present invention, two control liquid formulations (control 1 andcontrol 2) were prepared using the components listed in Table 7, below,and compared with human growth hormone (hGH) liquid formulationsprepared according to the same method as in Example 1.

TABLE 7 Major Isotonic component Buffer agent Surfactant StabalizerPreservative Solution Control 1 Somatropin Sodium D-mannito 1 Tween 20 —Benzyl Injectable 2.7 mg acetate alcohol solution 1 ml Contol 2Somatropin — D-mannito 1 Poloxamer Histidine Phenol Injectable 3.3 mg188 solution 1 ml DWF15 Somatropin Sodium D-mannito Macrogol-1 PEGBenzyl Injectable 2.5 mg acetate 135 mg 5 poly- 3001% alcohol 2.5 mgsolution 1 ml 10 mM oxystearate v/v 2 mg DWF16 Somatropin SodiumD-mannito Macrogol-1 PEG Benzyl Injectable 5.0 mg acetate 130 mg 5 poly-4001% alcohol 2.5 mg solution 1 ml 10 mM oxystearate v/v 2 mg DWF17Somatropin Sodium D-mannito Poloxamer L-Lys•HCl 1 mg Benzyl Injectable2.5 mg acetate 138 mg 188 2 mg alcohol 2.5 mg solution 1 ml 10 mM DWF18Somatropin Sodium D-mannito Poloxamer L-Lys•HCl 2 mg Benzyl Injectable5.0 mg acetate 138 mg 188 2 mg alcohol 2.5 mg solution 1 ml 10 mM DWF19Somatropin Sodium D-mannito Poloxamer L-Lys•HCl 1 mg Benzyl Injectable2.5 mg acetate 138 mg 188 2 mg alcohol 2.5 mg solution 1 ml 10 mM DWF20Somatropin Sodium D-mannito Poloxamer L-Lys•HCl 2 mg Benzyl Injectable5.0 mg acetate 138 mg 188 2 mg alcohol 2.5 mg solution 1 ml 10 mM

The hGH liquid formulations thus prepared were stored for 14 days underconditions of 40° C. and 75% RH. Then, the residual rates of somatropinwere determined using SEC-HPLC. Also, the formulations were incubated at2° C. to 8° C. for 24 hours with horizontal agitation (20 mm inamplitude, 220 oscillations per min), and were then examined foraggregation and appearance. The results are given in Table 8, below.

TABLE 8 40° C., 75% RH Agitation for 24 hrs Aggregation Aggregation Day14 Day 14 (somatropin %) (somatropin %) Appearance Control 1 97.00 ±0.15 94.57 ± 0.05 ◯◯ Control 2 99.11 ± 0.05 96.26 ± 0.07 ◯ DWF15 99.99 ±0.01 99.99 ± 0.01 X DWF16 98.22 ± 0.06 97.01 ± 0.15 ◯ DWF17 100.00 ±0.00  100.00 ± 0.00  X DWF18 99.98 ± 0.01 99.97 ± 0.02 X DWF19 100.00 ±0.00  100.00 ± 0.00  X DWF20 100.00 ± 0.00  99.98 ± 0.01 X Clear andtransparent (X) Slightly produced (Δ) Turbid (◯) Very High aggregateproduction (◯◯)

As shown in Table 8, aggregates were produced at the highest level inControl 1, which did not contain any stabilizer, and at the secondhighest level in Control 2, which was prepared using histidine as astabilizer. In contrast, the liquid formulations of the presentinvention, with the exception of DWF16, never produced aggregates. Theseresults indicate that a stable liquid formulation of human growthhormone may be prepared by combining a specific amino acid, L-lysine orL-arginine, or PEG 300, which serves as a stabilizer, with a specificsurfactant.

In addition, the liquid formulations were examined for the residualrates of somatropin when stored at 25° C. for three months and at 2° C.to 8° C. for up to one year. The results are given in Table 9, below,and FIGS. 1 and 2.

TABLE 9 25° C. (3 months) 2° C. to 8° C. (1 year) DeamidationAggregation Deamidation Aggregation (somatropin (somatropin (somatropin(somatropin %) %) %) %) DWF15 92.34 ± 0.20 98.24 ± 0.08 94.56 ± 0.1299.78 ± 0.02 DWF16 93.15 ± 0.15 98.45 ± 0.06 95.64 ± 0.05 99.43 ± 0.03DWF17 95.26 ± 0.19 100.00 ± 0.00  98.06 ± 0.05 100.00 ± 0.00  DWF1894.56 ± 0.18 99.93 ± 0.03 99.17 ± 0.09 100.00 ± 0.00 

As shown in Table 9 and FIGS. 1 and 2, the liquid formulations werestable during storage for a period of at least one year.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the present invention employed a specificstabilizer in combination with a specific surfactant in order toovercome the problem of low stability of a liquid formulation of humangrowth hormone, thereby suppressing the formation of deamidated productsof human growth hormone and, in particular, preventing aggregation intodimers or polymers, leading to remarkably enhanced stability. Thus, theliquid formulation according to the present invention is highlyresistant to external stresses during transport and storage.

1. A liquid formulation comprising human growth hormone; L-lysine or L-arginine; and poly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate or polyethylene glycol-35 castor oil.
 2. The liquid formulation according to claim 1, wherein the L-lysine or L-arginine is a hydrochloride salt.
 3. The liquid formulation according to claim 1, wherein the poly(oxyethylene) poly(oxypropylene) copolymer is poloxamer (poly(oxyethylene) poly(oxypropylene) copolymer)
 188. 4. The liquid formulation according to claim 1, wherein the human growth hormone is present in an amount ranging from 2.5 to 5.5 mg/ml; the L-lysine or L-arginine in an amount ranging from 0.02 to 0.5% (w/v) per mg of the human growth hormone; and the poly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate or polyethylene glycol-35 castor oil in an amount ranging from 0.1 to 0.5% (w/v).
 5. The liquid formulation according to claim 1, further comprising at least one selected from among a buffer, an isotonic agent, a preservative and an analgesic.
 6. The liquid formulation according to claim 5, wherein the human growth hormone is present in an amount ranging from 2.5 to 5.5 mg/ml; the L-lysine or L-arginine in an amount ranging from 0.02 to 0.5% (w/v) per mg of the human growth hormone; the poly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate or polyethylene glycol-35 castor oil in an amount ranging from 0.1 to 0.5% (w/v); sodium citrate or sodium acetate in an amount ranging from 5 to 20 mM; D-mannitol in an amount ranging from 20 to 50 mg/ml; and benzyl alcohol in an amount ranging from 1 to 9 mg/ml.
 7. A liquid formulation comprising human growth hormone; polyethylene glycol 300; and poly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate or polyethylene glycol-35 castor oil.
 8. The liquid formulation according to claim 7, wherein the poly(oxyethylene) poly(oxypropylene) copolymer is poloxamer (poly(oxyethylene) poly(oxypropylene) copolymer)
 188. 9. The liquid formulation according to claim 7, wherein the human growth hormone is present in an amount ranging from 2.5 to 5.5 mg/ml; the polyethylene glycol 300 in an amount ranging from 0.2 to 1.0% (v/v) per mg of the human growth hormone; and the poly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate or polyethylene glycol-35 castor oil in an amount ranging from 0.1 to 0.5% (w/v).
 10. The liquid formulation according to claim 7, further comprising at least one selected from among a buffer, an isotonic agent, a preservative and an analgesic.
 11. The liquid formulation according to claim 10, wherein the human growth hormone is present in an amount ranging from 2.5 to 5.5 mg/ml; the polyethylene glycol 300 in an amount ranging from 0.2 to 1.0% (v/v) per mg of the human growth hormone; the poly(oxyethylene) poly(oxypropylene) copolymer, polyethylene glycol-15 polyoxystearate or polyethylene glycol-35 castor oil in an amount ranging from 0.1 to 0.5% (w/v); sodium citrate or sodium acetate in an amount ranging from 5 to 20 mM; D-mannitol in an amount ranging from 20 to 50 mg/ml; and benzyl alcohol in an amount ranging from 1 to 9 mg/ml. 